Diabetes severely impairs male reproduction. The present study assessed the effects and mechanisms of action of advanced glycation end products (AGEs), which play an important role in the development of diabetes complications, on testosterone secretion by rat Leydig cells. Primary rat Leydig cells were cultured and treated with AGEs (25, 50, 100 and 200 µg/ml). Testosterone production induced by human chorionic gonadotropin (hCG) was determined by ELISA. The mRNA and protein expression levels of steroidogenic acute regulatory protein (StAR), cholesterol side-chain cleavage enzyme (P450scc) and 3β-hydroxysteroid dehydrogenase (3β-HSD), which are involved in testosterone biosynthesis, were measured by reverse transcription-quantitative PCR and western blot analyssi, respectively. Reactive oxygen species (ROS) production in Leydig cells was measured using the dichlorofluorescein diacetate (DCFH-DA) probe. The expression levels of endoplasmic reticulum stress-related proteins [C/EBP homologous protein (CHOP) and glucose-regulated protein 78 (GRP78)] in the Leydig cells were measured by western blot analysis. We found that the AGEs markedly suppressed testosterone production by rat Leydig cells which was induced by hCG in a concentration-dependent manner compared with the control (P<0.01). The mRNA and protein expression levels of StAR, 3β-HSD and P450scc were downregulated by the AGEs in a dose-dependent manner compared with the control (P<0.01). The antioxidant agent, N-acetyl‑L‑cysteine (NAC), and the endoplasmic reticulum stress inhibitor, tauroursodeoxycholic acid (TUDCA), reversed the inhibitory effects of AGEs. In addition, the content of ROS in Leydig cells treated with AGEs increased significantly. The expression levels of CHOP and GRP78 were markedly upregulated by the AGEs in the Leydig cells. From these findings, it can be concluded that AGEs inhibit testosterone production by rat Leydig cells by inducing oxidative stress and endoplasmic reticulum stress.

To understand the relationship between epidermal growth factor receptor (EGFR) and axon regeneration and the mechanisms of how EGFR regulates the neuronal intrinsic regenerative ability, we evaluated the levels of mRNA and protein of EGFR、total mammalian target of rapamycin (mTOR), p-mTOR(Ser2448) , total Akt and p-Akt(Ser473) in rats of different developmental stage by using Western blot and real-time polymerase chain reaction analysis. Axon protein tau and neuron proteins β-tubulin/neurofilament (NF) were assessed to evaluate the extent of the axon regeneration in cultured neuron cells. Expressions of EGFR、total mTOR, p-mTOR(Ser2448) , total Akt and p-Akt(Ser473) in cultured neuron cells were also detected using Western blot analysis. Our results showed that the expressions of EGFR and mTOR dropped off with the ageing of the rats, and Ser473 phosphorylation of Akt and Ser2448 phosphorylation of mTOR were highly expressed in foetal and newborn rats but decreased obviously in adult rats. tau, β-tubulin and NF were upregulated when EGFR was overexpressed and down-regulated after EGFR was blocked. The phosphorylation of mTOR and Akt was apparently elevated when EGFR was overexpressed and decreased when EGFR was blocked, which suggested that EGFR has the potential to regulate the neuronal intrinsic regeneration and mTOR and PI3K/Akt pathway activation may have an important role in it.

The extract of Broussonetia papyrifera has been proved to have antitumor activity. However, the underlying mechanism remains unclear. This study aimed to elucidate the mechanism of apoptosis of HepG2 cells induced by polyphenols from Broussonetia papyrifera (PBPs). The results revealed that PBPs inhibited the proliferation of HepG2 cells in a dose-dependent and time-dependent manner. Flow cytometry analysis showed that PBPs increased the apoptosis ratio of HepG2 cells significantly. PBPs increased intracellular reactive oxygen species (ROS) production and decreased intracellular superoxide dismutase (SOD) level of HepG2 cells. PBPs induced cell cycle arrest at G1 phase. Western blotting showed that PBPs upregulated the ratio of Bax/Bcl-2 and the expression level of Caspase-3, and activated p53 in HepG2 cells. The inhibition of proliferative relative signals (protein kinase B, PKB/AKT) and survival relative signals (extracellular signal-regulated kinase, ERK) were also observed in PBP-treated HepG2 cells. Our findings suggest that apoptosis of HepG2 cells induced by PBPs is mitochondria-mediated via inactivation of ERK and AKT signaling pathways.